Comparative research on cellular immunostimulation by whole-body irradiation]

The effect of the cellular immune response by total body irradiation was investigated. The transplant survival (skin grafts) was determined as immune parameter. Donors were colony-bred Wistar rats and recipients were colony-bred Sprague-Dawley rats. The investigations were carried out with irradiated rats and with rats irradiated after thymectomy and/or adrenalectomy as well as with animals without irradiation. A single total-body irradiation (1 and 2 Gy) was administered. The skin graft survival in irradiated rats was significant shorter (radiogenic immunostimulation) than in unirradiated rats; there were no significant differences between the operated (thymectomy and/or adrenalectomy) and not operated animals. Including precedent examinations this radiogenic immunostimulation is caused by a relatively selective inactivation of T-suppressor cells.     

Paracetamol inhibits replicative DNA synthesis and induces sister chromatid exchange and chromosomal aberrations by inhibition of ribonucleotide reductase

Effects of paracetamol have been studied in a hydroxyurea (HU)-resistant mouse mammary tumour cell line TA3H2, shown to overproduce the small subunit of ribonucleotide reductase. These TA3H2 cells were much more resistant than the TA3H (wild-type) cells towards the inhibitory effect of paracetamol on cell growth, IC50 0.55 mM paracetamol for the wild-type compared to 2.7 mM for the HU-resistant cells. The reduced cell growth was due to an inhibition of replicative DNA synthesis, judged from an increased percentage of cells in S-phase measured by flow cytometry. Furthermore, in the wild-type cells, the increase in the number of cells in S phase was already observed at 0.1 mM while in the HU-resistant cell line this effect was first seen at 3.0 mM paracetamol. HU inhibits ribonucleotide reductase by destroying a tyrosyl free radical located on the small subunit of the enzyme. By electron paramagnetic resonance we demonstrate that paracetamol added to crude cell extracts of HU-resistant cells also immediately destroys this radical. These results show that paracetamol reduces DNA synthesis by a specific inhibition of ribonucleotide reductase. A concentration-dependent induction of sister chromatid exchanges was found both with paracetamol (1.0-10 mM) and HU (0.3-3 mM) in wild-type cells whereas no such increase was observed in HU-resistant cells. Paracetamol (1 mM for 2 h) also increased the number of chromosomal aberrations CAs in wild-type cells (i.e. chromatid breaks and chromatid exchanges). The frequency of CAs was not increased in HU-resistant cells at paracetamol concentrations up to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis

Antineutrophil cytoplasmic antibodies (ANCA) of IgG class have been described at high prevalence in autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). Data on IgA class ANCA in these diseases are limited. The aim of this study was to determine the prevalence and fluorescence patterns of IgA class ANCA in AIH and PSC and to examine a relationship between the presence of IgA ANCA and clinical characteristics in these patients. Sera from 35 patients with PSC (21 with concomitant inflammatory bowel disease), 40 patients with AIH and 10 healthy controls were studied. ANCA were detected on ethanol-fixed neutrophils using an indirect immunofluorescence technique. ANCA of the IgA class were found in 20% of sera from patients with PSC and in 50% of AIH sera. The majority of AIH patients with IgA class ANCA showed a 'classical' perinuclear staining pattern, whereas the 'classical' and 'atypical' perinuclear fluorescence patterns were distributed equally in PSC. In sera containing IgG and IgA class ANCA simultaneously, IgG class ANCA showed an 'atypical' pANCA fluorescence pattern whereas IgA class ANCA produced a 'classical' perinuclear staining. The presence of IgA class ANCA was not associated with disease-specific clinical characteristics. IgA class ANCA are more frequently detected in sera of patients with AIH than PSC. The diversity of fluorescence patterns points to different target antigens of IgA class ANCA with distinct subcellular localizations.     

Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis

The sequential treatment of young Wistar rats with two differentcarcinogens (diethylnitrosamine - plus partial hepatectomy -as an initiator, and 2-acetylaminofluorene as a cytotometricselection pressure) induces the appearance of foci and nodulesof liver cells which are phenotypically altered. By means ofan algorithm which takes into account binuclearity as well ascell-to-cell aggregation it is possible to compute cellularploidy distributions from flow-cytometric analysis of eitherhepatocyte suspensions or suspensions of hepatocytic nuclei.Cell suspensions isolated from carcinogen-treated rats can beshown to contain, already after 8 weeks, 70% small, diploidhepatocytes, whereas suspensions from normal or partially hepatectomizedcontrol livers contain only 10% diploid cells (the remainder-beingmostly tetraploid). Isolated nodules, i.e., expanding clonesof proliferating cells, believed to be neoplastic precursorlesions, contained almost only diploid cells. These observationssuggest that the selective outgrowth of a population of small,diploid hepatocytes may be a significant early step in the developmentof liver cancer.     

Proliferation of diploid hepatocytes and nonparenchymal (oval) cells during rat-liver regeneration in the presence of 2-acetylaminofluorene

Treatment of rats with the carcinogen 2-acetylaminofluorene (2-AAF) during liver regeneration (Solt-Farber protocol) induced a selective outgrowth of diploid, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes (3-4 times increase) as well as of nonparenchymal (oval) liver cells. After cessation of treatment the oval cells rapidly disappeared, while the population of diploid, GGT-positive hepatocytes declined more slowly over the subsequent ten weeks. In animals pretreated with the initiating carcinogen diethylnitrosamine (DEN) a large fraction of the diploid, GGT-positive hepatocytes persisted. The results differ from those obtained with our standard, sequential treatment protocol (2-AAF given after completed regeneration), where there is no hyperproliferation of oval cells and where GGT-positive hepatocytes are found only in DEN-pretreated animals (Saeter et al, Carcinogenesis 9: 581-587, 1988). Different experimental models of liver carcinogenesis may thus present different patterns of liver cell proliferation, which should be taken into account when general hypotheses on the cellular origin of liver cancer are proposed.     

Model system for isolation of competent ovarian-carcinoma cells from fresh tumor-tissue by a magnetic separation technique (macs)

Flow cytometric analysis of tumor cells in carcinomas is hampered by the presence of a variety of different cells in the tumor tissue and the surrounding stroma. To obtain single competent tumor cells, we have established a model system which can be applied to separate living cells from fresh ovarian carcinoma tissue. Due to the lack of tumor-cell surface specific antibodies, we isolated tumor cells by a procedure called 'negative tumor cell selection'. For this purpose, fresh ovarian carcinoma tissue, immediately after surgery, was subjected to mechanical disintegration using an automated mincing device to obtain a single-cell suspension (approximately 10(7) cells/g). Collagenase D (0.005%) was added to prevent further aggregation. Cells other than tumor cells were then labeled with a set of monoclonal antibodies directed to cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells) and 5B5 (fibroblasts). Anti-isotype antibodies coupled to ferrit microbeads were then reacted with the cell suspension and those cells reacting with the microbeads retained on a steel wool matrix in a magnetic field (1). Tumor cells not reacting with the microbeads were recovered by a simple wash of the steel wool matrix. All incubation steps were at 4 degrees C. This procedure, which takes about 2 hours, enables fast and simple isolation of single, living competent tumor cells from fresh tumor tissue and also from ascitic or pleuritic effusions. In a model system with cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was about 93% and about 97% when re-subjected to the same procedure (respective recovery rates 75% and 50%). The still unlabeled tumor cells can subsequently be analyzed by flow cytometry or by central laser scanning microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Moreover, after detergent treatment and fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens as well as nuclear DNA staining for ploidy and S-phase determination becomes possible.     

Treatment of candidal infections with fluconazole in neonates and infants

To study the therapy, efficacy and safety of fluconazole in candidal mycoses during neonatal phase and infancy a case review in 53 newborns and infants was performed. The majority of these patients were premature with a median birth weight of 1120 g and born within gestational week 23-38. The median age at the onset of fluconazole treatment was 5 weeks. All patients had underlying diseases and several risk factors, which favored the occurence of a systemic candidal mycosis. Systemic candidiasis was the most frequent diagnosis (75.5%). Fluconazole was administered at a daily dosage of 5-6 mg/kg for a median duration of 21 days. The hepatic, renal and hematologic functions were assessed before, one, two, and three weeks after start of treatment. Yeasts were identified in 37 patients. The most common fungus isolated at baseline was Candida albicans (68%). Clinical cure or improvement was reported in 31 out of 38 patients (81.6%). Mycological cure was achieved in 25 out of 32 newborns and infants. Despite the limited number of patients with outcome data, these preliminary results of a small cohort clearly indicate the effective antifungal therapy with fluconazole in neonates and infants. No serious side effects were observed in fluconazole-treated patients. Two patients with megaureter-megacystis-hydronephrosis syndrome and severe meningoencephalitis showed a mild increase in liver enzymes. - Conclusion: Fluconazole seems to be an effective therapy for systemic and other forms of candidiasis in infants including very low birth weight infants (VLBWI; <1500 g). These favorable safety and efficacy data are similar to results obtained with fluconazole in older children and adults. These findings, however, must be supported by larger trials. The recommended daily dose is 5 mg/kg body weight. Only in VLBWI the dosage interval within the first two weeks of life should be prolonged up to 3 days and fluconazole serum levels should be monitored.